Molecular characterization of isolated lactic acid bacteria from different traditional dairy products of tribes in the fars province, Iran

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The present researchaimed to investigate the morphological, biochemical and genotypic characterization of dominant lactic acid bacteria that were isolated from traditional dairy products in Iranian tribes . A total of 75 samples of traditional yogurt, doogh from dairy units in Fars province were randomly collected. Isolation of lactic acid bacteria, biochemical and genotypic identification were conducted. Totally, 157 LAB isolates were selected.

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Nội dung Text: Molecular characterization of isolated lactic acid bacteria from different traditional dairy products of tribes in the fars province, Iran

MOLECULAR CHARACTERIZATION OF ISOLATED LACTIC ACID BACTERIA FROM DIFFERENT TRADITIONAL DAIRY PRODUCTS OF TRIBES IN THE FARS PROVINCE, IRAN Maryam Jafari1, Nabi Shariatifar1, Gholamreza Jahed Khaniki*1,Mehran sayadi**2, Abbas Abdollahi3 Address(es): Gholamreza Jahed Khaniki, Mehran Sayadi 1 Department of Food Safety and Hygiene, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. 2 Department of Food Safety and Hygiene, Faculty of Health, Fasa University of Medical Sciences, Fasa, Iran 3 Department of Microbiology, School of Medicine, Fasa University of Medical Sciences, Fasa, Iran. *Corresponding author: Ghjahedkh@yahoo.com , Mehransayadi6262@yahoo.com https://doi.org/10.15414/jmbfs.3621 ARTICLE INFO ABSTRACT Received 22. 8. 2020 Various traditional dairy products could be considered as an abundant source for Isolation/collection of new lactic acid bacteria (LAB) Revised 3. 5. 2021 with unique characteristics. The present researchaimed to investigate the morphological, biochemical and genotypic characterization of Accepted 11. 5. 2021 dominant lactic acid bacteria that were isolated from traditional dairy products in Iranian tribes . A total of 75 samples of traditional Published 1. 10. 2021 yogurt, doogh from dairy units in Fars province were randomly collected. Isolation of lactic acid bacteria, biochemical and genotypic identification were conducted. Totally, 157 LAB isolates were selected. Cocci and rod shape LAB were 53.50% and 46.49%, respectively. Biochemical tests showed the occurrence of 40.6% of the strains were Streptococcus, 2.25% of the strains were enterococci Regular article and 20.3% of the strains were lactococci. Most isolated lactobacilli were related to Lactobacillus plantarum and Lactobacillus bulgaricus. Genetically the presence of the following species was verified: Lactococcus lactis subsp. cremoris, Lactobacillus plantarum, Streptococcus thermophilus, Lactobacillus fermentum, Lactococcus lactis subsp. lactis, Lactobacillus bulgaricus, Lactobacillus helveticus, and Lactobacillus casei. The current study showed that thestrains which were isolated from traditional dairy products were not only appropriatefor use as starter adjuncts or cultures, but also they may provide a valuable gene pool for research and production of commercial starters with specific traits. Keywords: Biochemical Identification; Genotypic Identification; Lactic acid bacteria; Strain selection; Traditional dairy products INTRODUCTION LABs that were isolated from traditional dairy products in some tribes in Fars province, Iran. Fermentation is the most economical old methods for food preservation and storage that maintaince quality for long period (Mashak, Sodagari, Mashak, & MATERIALS AND METHODS Niknafs, 2014). Fermentation of lactic acid is a known method for producingseveral dairy products, such as yogurt, doogh ( savory yogurt-based Sample collection beverage), kashk (drained yogurt product), gharaghooroot (a nonfat-diary product after boiling Doogh and yogurt), cheese, etc (Abd El Gawad, Abd El Fatah, & 75 traditional doogh and yogurt sampleswere gleaned randomly from local areas Al Rubayyi, 2010; Azadnia & Khan Nazer, 2009). Traditionally, doogh is of Fars in Iran and were sampled by transferring 10 ml into in sterile plastic bags referred to a drinking product which is produced from yogurt dilution following a maintained at 4°C and analyzed within 24 h. After being transferredunder strong agitation stage in special waterproof sacs made from sheep or goat skin, refrigerated conditions (4°C) to the laboratory, the collected samples underwent called ‘Mashk’.(Kirdar, 2012; Noori, Keshavarzian, Mahmoudi, Yousefi, & microbiological analysis during the next 24 h. Nateghi, 2013) Produced centuries ago in Bulgaria, yogurt is a semisolid fermented product whichis currently being consumed in different countries. Lactic Acid Bacteria Isolation Yogurt is a dairy product which is generated byStreptococcus salivarius subsp. thermophilus (S. thermophilus) and Lactobacillus delbrueckii subsp. bulgaricus In completely sterile conditions, the collected samples were homogenized (L. bulgaricus), two bacteria which convert lactose into lactic acid (Çelik, 2007; andserially diluted in Ringer's solution and the aliquots (100 μl) of each dilution Tamime & Marshall, 1997). The existence of living organisms in fermented were spread-plated for the isolation of LAB. Plate Count Agar (PCA) (Merck, food is well known and many scientific research were done regarding the Germany) was employed to improve the isolation of LAB by incubating perti traditional fermented products. The microorganisms present in food mostly dishes at a temperature of 30°C for 72h. De Man Rogosa Sharpe (MRS) agar (pH specify the propertiesof the fermented food(flavor, texture, and 5.7,Merck, Germany) was utilized to isolatelactobacilli, while GM17 agar(pH acidity)andprovide several nutritional benefits. These organisms exist as food 7.15,Himedia, Hindi) was used for the isolation of streptococci, lactococci, and natural microflora or resultfrom adding starter cultures in food fermentation enterococci. The culture media were supplemented with 50 mg L-1 of natamycin (Abdi, Sheikh-Zeinoddin, & Soleimanian-Zad, 2006).Dairy starter cultures are to stop themold and yeasts from growing(Botes, Todorov, Von Mollendorff, vital to the production ofhigh quality and safeproducts in the modern dairy Botha, & Dicks, 2007). Through the use of the gas pack system, MRS plates industry.Starter cultures are utilized as single strain, mixed strain, or multiple were incubated in aerobical and anaerobical conditions (Merck Anaerocult Type strains depending on the product type (El Soda, Ahmed, Omran, Osman, & A) at 37°C for 48-72 h, and GM17 plates were incubated under the Morsi, 2003). Lactic acid bacteria (LAB) are frequently known as dairy starter sameconditions at 30°C and 42°C for 24-48h (Jokovic et al., 2008). culture which are used in various fermented milk products (Moulay et al., 2006). Morphologically distinct colonies from PCA, GM17 and MRS agar plates were LABs widely exist as indigenous raw milk microflora in nature. These randomly-picked, sub-cultured and purified by streak plating via the same microorganisms are Gram-positive, catalase-negative andhaveanimportant part in medium and were incubated at 37 C ̊ for 72 h;subsequently, the strains were food fermentation (Maqsood, Hasan, & Masud, 2013). The objective of the maintained at 4°C and refreshed by streaked every 2 weeks. Pure strains were current research was to studythe phenotypic and genotypic diversity of dominant further tested forcatalase production, gram staining, spore formation, cell 1
J Microbiol Biotech Food Sci / Jafari et al. 2021 : 11 (2) e3621 morphology, and oxidase activity. Catalase negative and Gram-positive, non- volume of three molar sodium acetate (pH 2.5) was added. Via adding 2 volumes spore forming, oxidase negative, cocci or rods isolates were selected as of ice cooled absolute ethanol, the nucleic acids were precipitated. Subsequently, presumptive LAB and stored in growth (MRS or GM17 or Tryptic soy broth the mixture underwent centrifugationat 14000 rpm for 10 min; after that,the (TSB)) medium containing 15% (vv-1) glycerol as stocks frozen at -80°C. supernatant was discarded and the created pellet was washed two times by 70% ethanol. The remaining ethanol was eliminated through drying the pelletat room Biochemical identification temperature. It was then dissolved in TE buffer and kept at -20°C. Amplification of 16S rRNA genes and specific PCR conditions Biochemical identification of the isolated/selected bacteria was carried out following Bergey’s manual of determinative bacteriology.(Breed, Murray, & The mixtures of the PCR reaction were prepared in 25 μl volumes containing 2.5 Smith, 1957) For biochemical identification, every single isolate was activated in μl of 10X Taq buffer, 1.5 μl MgCl2 (50 mM), 2.5 μl dNTPs (2 mM/μl), 2 μl 5 ml GM17 or MRS or TSB broth medium for 24 h at 37°C prior to use. The tests random primer (16S-FA 5´-AGAGTTTGATCCTGGCTCAG-3´and 16S-RA 5´- used for preliminary characterization of isolates included Gram staining, catalase AGGAGGTGZTCCAGCCGC-3´), 0.5 μl Taq-DNA polymerase (5 U/μl). test (H2O2, 3%), oxidase test and endospore formation(spore staining). Isolates Through adding sterilized distilled water, the mixture was diluted up to 25 μl . with Gram-positive, catalase, and oxidase-negative, non spore forming The amplifications were carried out via an Applied Biometra thermalcycler characteristics were selected for further identification (Nikita & Hemangi, programmed to repeat the thermal profile.The PCR program included 5 min at 2012). 96°C; 30 cycles of 96°C for 30 s, 55°C for 30 s and 72°C for 30 s;another extension step at 72°C for 5 min was also performed in a mastercycler. Using Identification of cocci electrophoresis, theamplification products were assessed. Afterwards, the standard marker and the DNA fragments wereseparated in 1.5% ethidium Inoculated bacterial isolates in GM17 broth medium were incubated at 10°C, bromide-stained agarose gels. Then, the separated fragments(1200-1500 bp) and 40°C and 45°C. At different NaCl concentrations (2%, 4% and 6.5%), the growth their patterns were visualized and photographed with gel documentation system ability was evaluated in the modified GM17 broth media. The change of the color (Terzic-Vidojevic et al., 2014). as the evidence for cell growth (Nikita & Hemangi, 2012). Growth at pH 9.6, was detected by cultivating of isolates in the GM17 broth media with pH 9.6 (by Multiple sequence alignment pH adjustment with NaOH). Ultimately, the amplified genes were obtained from the gels by use of a QIA Arginine hydrolysis and gas production from citrate and Reduction of quick gel extraction kit (Bioneer, USA) according to the the manufacturer’s Methylene Blue instructions. Buffer 1 (gel binding buffer) was added to the cutted piece of gel, incubated for 10 min at 60°C to completely desolve the gel. The solution was Inverted Durham tubes and Reddy broth were applied in this test. The color of transferred to the DNA binding column tube, centrifuged for 1 min at 13000 rpm cultures usingargininechanged from yellow to violet. To reduce Methylene Blue, and poured in the flow-through, re-assembling the DNA binding filter column overnight cultures were transferred into GM17 broth tubes with Methylene Blue. with 2.0 ml collection tube. 500 μl of buffer 2 (cell lysis buffer) was added to the DNA binding column tube, centrifuged for 1 min at 13000 rpm., poured in Carbohydrate fermentations thethrough, re-assembling the DNA binding filter column with the 2.0 ml collection tube and dried via another centrifugation for 1 min at 13000 rpm. DNA The properties of the isolates were further determinedaccording to the profiles was eluted from column by adding 30 μl buffer 3 (EB) to the center of the DNA related to their sugar fermentation. Sixteen different sugars were utilized. In each binding filter column and remaining for at least 1 min at room temperature and test, the strains were inoculated in 3 ml of Phenol red base broth media centrifugation 1 min at 13000 rpm. The purified DNA fragments of amplified containing 150 µl of filter sterilized (0,22 µm, Merck Millipore) 10% sugar genes for 16S RNA were finally subjected to sequencing service (Macrogen, solutions (0.5%). The acidification as a measure of fermentation ability is South Korea) for standard sequencing (Poormontaseri, Hosseinzadeh, & reflected in color change from red to yellow. Shekarforoush, 2014). Identification of lactobacilli DNA sequence Analysis Inverted Durham tubes and MRS broths were usedto identify lactobacilli. The DNA sequences were obtained by use of forward and reverse primers assembled accumulation of gas in Durham tubes indicatedthe production of CO2 from and edited through the use of BioEdit sequence alignment editor version 5.0.9. glucose. Lactobacilli isolates were screened to determine if they were ableto Using BLAST, the homology was searched in the Genbank DNA database, and fermentD (-) Raffinose, D Mannitol, D (+) Galactose, D (-) Ribose, Lactose, the sequence similarity was estimated. Maltose, , glucose, L (+) Arabinose, D (-) Salicin, D (+) Xylose, D (+) Mannose, Fructose sucrose, Sorbitol, Trehalose, and Rhamnose as described above. RESULTS AND DISCUSSION Genotypic identification Lactic Acid Bacteria Isolation DNA extraction From the collected samples, approximately 280 colonies were picked at random from selective media for each group of LAB. To extract the DNA, the cultures of isolates grown in MRS and GM17 broth for 18h were employed. Genomic DNA extraction was donethrough phenol- Phenotypic Identification chloroform extraction, which is a modified bacterial DNA extraction protocol depicted by Federici et al.,and the samples of DNA were maintained at -20°C Selected colonies from the fermented food samples showed comparable (Federici et al., 2014). A single colony of each isolate was inoculated into 10 ml differences in their colony morphology (size, shape, shine and color). Among the of the appropriate medium broth and incubated overnight at 37°C. Using 280 Gram-positive isolates that were extractedfrom the dairy products, 117 (all centrifugation at 7500-8000 rpm for 5 min, the cells were harvested, and genomic belonging to the LAB family) were negative for catalase and oxidase activity DNA was isolated based on a modified genomic DNA isolation protocol andnon-spore forming bacteria . A large number ofisolates (60 isolates) had a rod (Ashmaig, Hasan, & El Gaali, 2009). The collected bacterial pellet was shape, therebypossibly related tothe genus Lactobacillus; the rest were cocci (57 resuspended in 2.5 ml of TE buffer (1M Tris-HCl, 0.5 mM EDTA pH 8.0) and isolates). washed two times with the same buffer. Once washed,the pellet was re- suspended into 250 μl of lyses solution (1% (w/v) SDS, 0.2 mol NaOH). Through Physiological and Biochemical Identification gentle shaking, the cell suspension was incubated for 1 h in a water bath at 65°C . The solution was gently emulsified by an equal amount of TE buffer saturated The results of identification of coccoid shaped isolates are shown in Table 1. phenolchloroform (1:1); it was thencentrifuged for 5 min at 7500-8000 rpm, and According to obtained results, the strains were Streptococcus (29 isolates), the aqueous phase was transferred intonew tubes. Next, the aqueous solution enterococci (12 isolates) and lactococci (16 isolates). The ability of lactobacilli waswashed two times with an equal amount of chloroform: isoamylalcohol, 24:1, isolates to ferment different types of carbohydrate is shown in Table 2. and it was centrifugated for 5 min at 7500-8000 rpm. Oncethe aqueous phase was centrifuged, it was transferred to Eppendorf tube (400 μl/ tube) to which1/10 2
J Microbiol Biotech Food Sci / Jafari et al. 2021 : 11 (2) e3621 Table 1 Technological properties of cocci shaped of cocci LAB that were isolated from dairy products and the fermentation profile of sugars. Lactococcus Lactococcus Streptococcus Entrococcus Enterococcus Entrococcus Characteristics lactis subsp. lactis subsp. thermophiles faecium fecalis durans lactis cremoris Gram staining + + + + + + catalase production - - - - - - oxidase activity - - - - - - spore formation Non - spore Non-spore Non-spore Non-spore Non-spore Non-spore Growth at pH 9.6 - + + + - - Reduction Methylene Blue - + + + ± - Growth at 10 °C - + + + + + Growth at 40 °C + + + + + - Growth at 45 °C + + + + - - Growth in 2% NaCl ± + + + + + Growth in 4% NaCl - + + + + - Growth in 6.5% NaCl - + + + - - Hydolysis of arginine - + + - + + CO2 from citrate - - - - Acid formed from Mannitol - ± + - ± - Raffinose - ± - - - - Ribose - + + + + - Galactose - + + + + + Maltose - + + + + - Lactose + + + + + + +: Growth / Fermentable carbohydrates - : Non-growth / Non-fermentable carbohydrates Table 2 Carbohydrates fermentation pattern of LAB isolated from traditional yogurt and doogh Lactobacillus species Characteristics Lb. Lb. Lb. Lb. Lb. casei Lb. Lb. Lb. Lb. plantarum delbruecki heleviticus brevis subsp. tolerans casei acidophilus bulgaricus salivarius Arabinose + - - + - - - - - Lactose + - + - + + + + + Glucose + + + + + + + + + Glucose (gas) - - - + - - - - - Xylose + - - + - - - - - Mannitol + - - - - + - - + Rhamnose - - - - - + - - + Salicin + - - - + + + - - Mannose + + - - + + + - + Ribose + - - + + + - - - Galactose + - + - + + - + + Sucrose + + - + - + + - + Raffinose + - - - - - + - + Fructose + + - + + + + + + Sorbitol + - - - + + - - + Trehalose + - + - - + + - + Lb: Lactobacillus, +: Growth / Fermentable carbohydrates, - : Non-growth / Non-fermentable carbohydrates Fig 1 shows identification results of the isolated LAB based on the phenotypic characterization. In addition, the results show that LAB isolates can be considered as the predominant bacteria in collected dairy products. Nonetheless, different dairy samples had various predominant LAB. Figure 1 The percentage of LAB species isolated from collected samples 3
J Microbiol Biotech Food Sci / Jafari et al. 2021 : 11 (2) e3621 In addition, we found that cocci especially those cultured by pouring plate, have subsp cremoris and 65 (55.56%) L. mesenteroides subsp cremoris among LAB complitely different forms, however with the aid of a microscope, they were seen cocci and regardingLAB bacilli ,L. helveticus 85 (15.3%); L. plantarum 124 as spherical or oval, mostly forming short or long chain and in single or pairs. (22.3%); L. brevis 117 (21%) ;L. casei subsp. casei 86 (15.5%) and L. delbruckii Bacilli were observed to be so differently from each other under a microscope; subsp. bulgaricus 144 (25.9%) were detected (Azadnia & Khan Nazer, 2009). although, on the plate, they were almost similar except L. bulgaricus with a star To investigate different species isolated from Iran using phenotypical and shape. Diversity of bacterial species can be related to different factors, such molecular tests, Tajabadi Ebrahimi et al. performed a study on traditional Iranian asraw milk composition and the breed of the animal,regional influences, the kind dairy products. Isolated species belonged to L. plantarum (cheese, fermented of the utilized culture medium, effect of reducing pH environment at different milk, yogurt, kashk), L. brevis (cheese, yogurt), L. casei (cheese, yogurt, stages of growth, concentration of salt in the environment, and the existenceof fermented milk), L. acidophilus (yogurt), L. jensenii (cheese), L. salivarius calcium(Kirdar, 2012; Rahimi et al., 2012; Wright & Klaenhammer, 1983). (yogurt), L. lactis (cheese), L. alimentarium (yogurt), L. rhamnosus (cheese), L. In order to classify cocci shaped isolates, growth ability at 10°C, 40°C, and 45°C arieminis (cheese), L. agilis (kashk) (Ebrahimi, Ouweh, Hejazi, & Jafari, as well as growth at 2%, 4% and at the consentration of 6.5% NaCl were used. 2011). Streptococcus thermophilus was not able to show growth at 10°C, but it had a Acurcio et al. (2014), García-Cano et al. (2019) and Masalam et al. (2018) good growth at 40°C and 45°C; 47 isolates showed these characteristics. noted that strains of Enterococcus, Lactococcus, Lactobacillus, Streptococcus Enterococcus species could grow at 10°C, 40°C and 45°C; 3 isolates showed and Weissella isolated from raw milk and dairy products have in vitro probiotic these characteristics. Unlike other species, Lactococcus species indicated growth features, including resistance to biliary salts and gastric juiceand antagonism at 10°C but had no growthat 45°C and only 26isolates possessed such property. against reference pathogens, includingEscherichia coli, Listeria monocytogenes, L. lactis subsp. lactis strain could grows poorly at 45°C, while L. lactis subsp. Staphylococcus aureus, Salmonella spp., and Shigella sonnei (Acurcio et al., cremoris grew just at 10°C. S. thermophilus and L. lactis subsp. cremoris did not 2014; Bin Masalam et al., 2018; García-Cano et al., 2019). The quality of have the ability to grow at salt concentrations of 4 and 6.5%, while L. lactis cheese and other fermented food products depends on the ability of subsp. lactis and Enterococcus species could grow at different levels of salt. microorganisms to produce flavour and aroma in the starter culture. Various Reduction methylene blue, growth at a pH of 9.6, arginine hydrolysis, and citrate LABs have been assessed to determine if they are ableto degrade amino acids utilization and carbohydrate fermentations also were examined. Only 11 isolates into aroma compounds. L.lactis subsp. lactis and L.lactis subsp. cremoris, were able to reduce methylene blue and hydrolyze the arginine. At a pH of 9.6, L.lactis, L. helveticus, L. bulgaricus and L.casei have this ability to very few (only 2 isolates) could grow and 5 of them were able to produce gas degrademethionine to methonethiol, dimethyledisulphide and dimethyltrisulphide from citrate. Cocci shape isolates capable of fermenting carbohydrates(raffinose, (Abiona & Adegoke, 2017). These strains, which have high lipolytic and mannitol, ribose, lactose, maltose, galactose)were also evaluated. S. thermophilus proteolytic activities were genetically identified and proved to playa crucial role and L. lactis subsp. cremoris bacteria had less ability to ferment carbohydrates, in cheese ripening (Abosereh, Abd El Ghani, Gomaa, & Fouad, 2016; García- while Enterococcus species and some species of Lactococcus are able to ferment Cano et al., 2019). majority of tested sugars. S. thermophilus and L. lactis subsp. cremoris bacteria differ in the fermentation of galactose; S. thermophilus poorly ferments galactose Genotypic identification because theits enzyme beta-galactosidase is not able to ferment this sugar. Similarly, most researchers agree on the fermentation of lactose by cocci bacteria Since 1990, some alternatives have emerged for the classical phenotypic and (Begovic et al., 2011; Patil, Pal, Anand, & Ramana, 2010). On the contrary, biochemical identification of LAB . These methods might not be sufficient to atypical characteristic of some Streptococcus strains is their galactose definitively attribute a strain to a certain species. To thoroughly identify and fermentation. Almost none of LAB cocci were able to ferment raffinose, classify the species in bacterial systematics, polyphasic approaches (Coeuret, however, they are able to break the disaccharide lactose into glucose and Dubernet, Bernardeau, Gueguen, & Vernoux, 2003), such asphenotypic, galactose that can be used for energy. chemotaxonomic, and genotyping methods are still recommended. For identification of bacilli shaped isolates, carbon dioxide from glucose and To identify the LAB species more accurately, PCR assays were used with carbohydrate fermentation tests were performed by applying 13 different type of primers that target 16S rRNA gene (Table 3). To confirm the belonging to certain carbohydrates. The biochemical tests showed that Lactobacillus species could be species, the nucleotide sequences of the 16S rRNA gene related to all the isolates divided to three groups. Group I comprises obligately homofermentative strains were examined and specified using the BLAST program on NCBI. The results (L. acidophilus, L. delbruckii, L. helveticus). The second category includes were categorized based onthe maximum identity andrecorded in terms of facultative heterofermentative strains (L. casei, L. plantarum), and group III are coverage. The sequence similarity with 90% or higher cut-off was regarded as obligately heterofermentative strains (L. brevis, L .fermentum); This means that significant.The optimum hit was considered as the sequence with the most for the fermentation of sugar, only the 6-PG/PK pathway is accessible. In order to maximum similarity to the query sequence. differentiate homofermentative and heterofermentative LAB, the ability of the isolates was studied in the fermentation of pentose sugars such as ribose and Table 3 Genotypes of the selected isolated LAB as 16S rDNA gene sequence xylose. All cocci isolates were able to ferment pentose in order to identify species alignments submitted to the NCBI GeneBank database. more accurately. This survey and similar studies indicate the approximate Species Similarities (%) Source of Isolation different sugars fermented by Lactobacillus species (Ayad, Nashat, El-Sadek, Entrococcus faecium 99% Doogh Metwaly, & El-Soda, 2004; Erkuş, 2007; Khedid, Faid, Mokhtari, Streptococcus termophilus 100% Yogurt Soulaymani, & Zinedine, 2009; Moulay et al., 2006; Omafuvbe & Enyioha, Lactococcus lactis subsp. lactis 99% Doogh 2011). Regarding galactose and mannose sugars ability fermentation by L. Entrococcus durans 100% Yogurt bulgaricus, contradictory findings were reported by researchers. Asmahan (2011), reported that L. delbrueckii is not capable of fermenting the mannose Lactococcus salivarius 95% Doogh sugar, while Kiaei et al. in his research reported that mannose was fermented by Lactobacillus heleviticus 98% Yogurt L. delbrueckii (Ali, 2011). Majority of researchers have reported in their studies Lactobacillus delbrueckii subsp. 100% Yogurt that Lactobacillus strains are not able to ferment galactose (Ashmaig et al., Bulgaricus 2009), but Maqsood et al. have suggested that L. bulgaricus can ferment the Lactobacillus plantarum 97% Yogurt galactose. L. delbueckii, L. bulgaricus and L. heleviticus possess weak Lactobacillus casei 98% Yogurt fermentation ability and are only able to ferment few suagrs (Maqsood et al., Lactobacillus acidophilus 95% Doogh 2013). In a previously reported research by Kirdar (2012) in meaditerian area, three Lactobacillus species were noted. Unlike to other lactobacilli, L. casei With the current rapid growth in biotechnology, more and more molecular subsp. pseudoplantarum is able to obtain energy and produce acid from most of techniques are utilizedin the genetic diversity studies on LAB. Typing methods the carbohydrates (Abdullah & Osman, 2010). The ability to ferment galactose based onmajor advantages because of their highly discriminatory power, being is considered as the main factor for distinction between L. helveticus species as applicable universally and having independent culture. Methods like pulsed field galactose positive and L. bulgaricus known as galactose negative. L. lactis and gele electrophoresis (PFGE), DNA/DNA hybridization, ribotyping, polymerase L. acidophilus ferment sugars in the same way, however, the only distinction chain reaction (PCR), real-time PCR hybridization and sequencing of rRNA are between them is in the fermentation of raffinose, in which L. lactis is not able to able to distinguish even the close related strains (Miljkovic et al. 2019; use the sugar for its energy supply. Roshanzade et al. reported the similar action Axelsson, 2004; Furet, Quénée, & Tailliez, 2004). of these two species in the fermentation of sugars (RoushanZadeh, Eskandari, In categorizing LABs, a large number of studies have compared the foregoing Shekarforoush, & Hosseini, 2014). Bacterial strains oxidize environmental genetic methods. A highly comprehensive study in this regardis still the energy sources by producing different enzymes; however, using energy sources collaborative work carried out by M. Sta°hl, M.-L. Johansson andcolleagues. depends on the synthesis of specific enzymes by each bacteria (Ashmaig et al., They made use of a set of Lb. reuteri and Lb. plantarum strains. These strains 2009). According to the literature, new bacterial species identified are discovered weresystematically characterized via phenotypic tests, DNA-DNA homology, in almost the same studies carried out previousely, pointing that the main reason REA, automated PCR sequencing of rRNA, ribotyping, and RAPD. Their work for diverzification could be adaptation to the local conditions of the region. clearly elucidates that every method has its own upsides and downsides,and that Biochemical tests for the representative LAB isolated from traditional yogurts in one technique cannot bethe answer to all problems, but rather different methods various parts of Fars province revealed the occurrence of 52 (44.44%) L. lactis complement one another. 4
J Microbiol Biotech Food Sci / Jafari et al. 2021 : 11 (2) e3621 CONCLUSION Coeuret, V., Dubernet, S., Bernardeau, M., Gueguen, M., & Vernoux, J. P. (2003). Isolation, characterisation and identification of lactobacilli focusing In the present study, in-vitro approaches were chosen to examine the diversity of mainly on cheeses and other dairy products. Le Lait, 83(4), 269-306. the LAB in dairy products from Iran. Previous studies have been conducted on https://doi.org/10.1051/lait:2003019 LAB isolates from the Iranian traditional cheeses and yogurts, with less attention Ebrahimi, M. T., Ouweh, A. C., Hejazi, M. A., & Jafari, P. (2011). Traditional to others traditional and industrial dairy products. This work showed that Iranian dairy products: A source of potential probiotic lactobacilli. African other fermented dairy products could be significant sources of LAB. Some journal of microbiology research, 5(1), 20-27. studies reflected the fact that LAB strains detected from dairy products, produce https://doi.org/10.5897/AJMR10.629 more pleasant flavor and volatile compounds than industrial strains. Local LABs El Soda, M., Ahmed, N., Omran, N., Osman, G., & Morsi, A. (2003). Isolation, are highly potential as starters or adjuncts. To optimally utilize novel strains, the identification and selection of lactic acid bacteria cultures for cheesemaking. specific conditions of use have to be definedand more detailed research must be Emirates Journal of Food and Agriculture, 51-71. conducted on their technological features. https://doi.org/10.9755/ejfa.v15i2.5006 Erkuş, O. (2007). Isolation, phenotypic and genotypic charecterization of yoghurt Acknowledgements: The present study was extracted from a thesis financed by starter bacteris. Izmir Institute of technology, the School of Public Health, Tehran University of Medical Sciences (grant no. 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